Although in genetic engineering techniques numerous procaryotic vector-host-systems for cloning of heterologous or homologous genes are already known, there is a continuous need for novel systems which may have advantages over the known systems.
Most recombinant work in DNA technology has been carried out with bacteria such as Escherichia coli or Bacillus subtilis. The lactic acid bacteria, however, are of much more industrial interest. For this reason, a number of efforts have been made to develop plasmid vectors for cloning and expression of homologous and heterologous genes in lactic acid bacteria, particularly in Lactobacillus spec. and Lactococeus spec. [see PCT WO 85/03945, Gasson and Anderson (1985), EP-A-0 316 677, Bates et al. (1989), Joset al. (1985) and review articles of Chassy (1987) and of De Vos (1987)]. Lactococcus spec. was formerly named Streptococcus spec.
Lactococcal strains investigated so far harbour a characteristic plasimid complement consisting of multiple different plasmids. This property can be used to differentiate between various lactococcal strains (Davies et al., 1981). For genetic studies, plasmid free strains have been constructed by repeated curing in the course of plasmid function studies (De Vos, 1987).
For example, the plasmid complement of L. lactis 712, hereinafter also called L. lactis LL712, consists of 5 plasmids having the molecular weights of 1.8 Md, 2.5 Md, 5.2 Md, 9 Md and 33 Md which are named pSH71, pSH72, pSH73, pSH74 and pLP712, respectively (Gasson, 1983).
Based on plasmid pSH71 of L. lactis and on the related L. cremoris plasmid pWV01 (Otto et al., 1982) various cloning vectors have been constructed. The cloning vectors have been produced either by inserting genetic markers such as antibiotic resistance genes into the plasmids or by screening fragments of the plasmids for an origin of replication function, i.e. for the ability to sustain replication of selected DNA fragments. A cloning vector produced according to the latter method is pNZ12 which contains the 1.7 kbp ClaI restriction fragment of pSH71 comprising the origin of replication (Gasson and Anderson, 1985). The origin of replication of pSH71 is also functional in other gram-positive bacteria such as Bacillus spec. and in the gram-negative Escherichia coli.
On the basis of these plasmids cloning vectors useful for the introduction and expression of homologous or heterologous genes in lactic acid bacteria have been developed. The development of the cloning vectors resulted in transformed lactococcal strains with improved properties which are useful in food and feed industry, for example a bacteriophage resistant L. lactis strain (EP-A-0 316 677) or a L. tactis strain which produces bovine prochymosin (PCT WO 85/03945). The development of cloning vectors for the production of homologous or heterologous gene products is not only of interest because of the production of improved lactic acid bacteria cells but also for the production of recombinant proteins. One of the major problems with the production of heterologous proteins in microbial expression systems has been the purification of the product. Purification of intracellular proteins is time-consuming and often results in poor yields. Purification can be considerably facilitated if the product is secreted from the host cell. To avoid the problems of purification of the products expressed in bacteria, vector-host systems for the production of recombinant proteins which are secreted into the supernatant can be useful.
Another advantage of secreted proteins can be that they can have a native and biologically active conformation, because then no refolding process is needed. Refolding is usually necessary if the polypeptide is intracellularly deposited.
Secretion of a protein usually requires a signal peptide at the amino terminus of the primary translation product which directs the protein into the secretory pathway. It is of advantage if the signal peptide is cleaved enzymatically from the protein during the translocation through the cell membrane. This, however, is not always the case.